7α- and 7β-Hydroxyorobanchyl acetate as germination stimulants for root parasitic weeds produced by cucumber

Pichit Khetkam; Xiaonan Xie; Takaya Kisugi; Hyun Il Kim; Kaori Yoneyama; Kenichi Uchida; Takao Yokota; Takahito Nomura; Koichi Yoneyama

doi:10.1584/jpestics.D14-038

Abstract

Cucumber (Cucumis sativus) plants were found to exude at least 12 germination stimulants for root parasitic weeds including 5 known strigolactones, 7-oxoorobanchol, 7-oxoorobanchyl acetate, orobanchol, orobanchyl acetate, and 4-deoxyorobanchol. Two novel germination stimulants were purified from cucumber root exudates and their structures were determined to be 7α- and 7β-hydroxyorobanchyl acetate by 1D and 2D NMR spectroscopy, and ESI- and EI-MS spectrometry. The stereochemistry was determined by NOE measurement and by comparing the CD spectra with those of the synthetic standards of four stereoisomers of orobanchol. 7α- and 7β-Hydroxyorobanchol were detected by LC-MS/MS, and GC-MS analysis of purified sample confirmed their structures. The germination stimulation activities of 7α- and 7β-hydroxyorobanchyl acetate on Orobanche minor were comparable to those of orobanchyl acetate and 7-oxoorobanchyl acetate. By contrast, 7β-hydroxyorobanchyl acetate was a highly potent germination stimulant for Phelipanche ramosa inducing more than 50% germination at 10 pM.

Introduction

Seed germination of root parasitic weeds including witchweeds (Striga spp.) and broomrapes (Orobanche spp. and Phelipanche spp.) is induced by germination stimulants produced by and released from host roots.^1,2) Various germination stimulants have been identified in the root exudates of host and non-host plants. So far, three different classes of compounds, dihydroquinones, sesquiterpene lactones and strigolactones (SLs), are identified as germination stimulants for root parasites.^2,3) Among them, SLs are the most potent stimulants, inducing germination at ≤10 pM.⁴⁾ SLs function not only as germination stimulants for root parasitic weeds but also as host recognition signals for symbiotic arbuscular mycorrhizal (AM) fungi in the rhizosphere.⁵⁾ In addition, SLs are a novel class of plant hormones involved in the regulation of shoot^6,7) and root architecture,^8,9) photomorphogenesis,¹⁰⁾ secondary growth,¹¹⁾ senescence,¹²⁾ and seed germination.¹³⁾

To date, more than 20 SLs have been isolated and characterized from root exudates of various plant species.¹⁴⁾ All natural SLs contain a tricyclic lactone (ABC part) that connects via an enol ether linkage to a butenolide moiety (D-ring) and carry different substituents on the A- and B-rings but the same C–D moiety.¹⁴⁾ Natural SLs can be divided into two groups, orobanchol-type SLs with an α-oriented C ring and strigol-type SLs with a β-oriented C-ring, while they all posses a 2′(R) configuration.^14–16) The biosynthesis of SLs has been proposed to start from β-carotene via a pathway mediated by the enzymes, D27, CCD7 and CCD8 to give an intermediate called carlactone.¹⁷⁾ The oxidation of C19, the methyl group of carlactone, and the ring closing reactions afford 5-deoxystrigol and its isomer 4-deoxyorobanchol (ent-2′-epi-5-deoxystrigol), common precursors for the other SLs.

In our earlier work, we detected two novel SLs in addition to 7-oxoorobanchol and its acetate in the root exudates from flax (Linum usitatissimum).¹⁸⁾ Although we extrapolated the structures of the two SLs as hydroxyorobanchyl acetates based on their LC-MS spectra and retention times in reversed phase (RP)-HPLC, we could not obtain enough pure compounds for NMR spectroscopy. In this study, we report on the isolation and structure elucidation of these novel SLs as 7α-hydroxyorobanchyl acetate (1) and 7β-hydroxyorobanchyl acetate (2) from cucumber (Cucumis sativus root exudates and their germination stimulation activity toward the two root parasitic weeds Orobanche minor and Phelipanche ramosa.

Fig. 1. The structures of strigolactones produced by cucumber plants; 7α-hydroxyorobanchyl acetate (1), 7β-hydroxyorobanchyl acetate (2), 7-oxoorobanchyl acetate (3), 7-oxoorobanchol (4), orobanchyl acetate (5), orobanchol (6), 4-deoxyorobanchol (7), 7α-hydroxyorobanchol (8) and 7β-hydroxyorobanchol (9).

Materials and Methods

1. Instruments

¹H and ¹³C NMR spectra were recorded in CDCl₃ (δ_H 7.26, δ_C 77.0) by a JEOL JMN-ECA-500 spectrometer. Standard pulse sequence and phase cycling were used for HMQC, HMBC, COSY and NOE spectral analyses. CD spectra were recorded with a JASCO J-720W spectropolarimeter in MeCN. EI/GC-MS spectra were obtained with a JOEL JMS-Q1000GC/K9 on a DB-5 (J&W Scientific, Agilent) capillary column (5 m×0.25 µm) using a He carrier gas (3 mL/min). GC-MS was operated according to Yokota et al.¹⁹⁾ with minor modifications.²⁰⁾ The column temperature was kept at 130°C for the first 1.5 min and elevated to 270°C by a 6°C/min gradient. High-resolution mass spectra (HR-MS) were obtained with an Agilent 6520 Q-TOF mass spectrometer equipped with an ESI source. ESI-LC-MS analyses were performed with a Quattro LC tandem MS instrument (Micromass, Manchester, UK). The LC-MS analytical conditions were essentially the same as those described previously.²¹⁾ The HPLC analysis and purification were conducted on a Hitachi L-7100 high-pressure gradient system equipped with a PDA detector. Silica gel column chromatography (CC) was performed on silica gel (Wakogel C-300, Wako Pure Chemical Industries, Ltd., Japan).

2. Chemicals

The optically pure stereoisomers of orobanchol, synthesized according to the reported method,²²⁾ were generous gifts of Professor Kohki Akiyama (Osaka Prefecture University, Japan). The other chemicals of analytical grade and HPLC solvents were obtained from Kanto Chemical Co., Ltd. and Wako Pure Chemical Industries, Ltd.

3. Plant material

Seeds of cucumber (C. sativus cv. Aonagakei-Jibai) were purchased from a local market. O. minor seeds were collected from mature plants that parasitized red clover (Trifolium pratense) grown in the Kinu basin of Tochigi Prefecture, Japan. P. ramosa seeds (pathotype 1, parasite of Brassica napus) were kindly provided by Professor Philippe Delavault (University of Nantes, France).

4. Seed germination assay

Germination assays of O. minor and P. ramosa seeds were conducted as reported previously.²¹⁾ Surface-sterilized seeds, ca. 30 each, were sown on 5-mm glass fiber disks (Whatman GF/A, Tokyo, Japan). Ninety disks were incubated in 9-cm Petri dishes each lined with a sheet of filter paper (Advantec No. 2, Tokyo, Japan) and wetted with 6 mL of Milli-Q water. The Petri dishes were sealed with Parafilm, wrapped in aluminum foil, and placed in the dark at 23°C for 7 days. Three disks carrying the conditioned seeds were transferred to 5-cm Petri dishes each lined with a sheet of filter paper and wetted with 650 µL of test solution. Each test solution, unless otherwise mentioned, contained 0.1% (v/v) MeCN. The Petri dishes were sealed, wrapped in aluminum foil, and incubated in the dark at 23°C for 4–5 days. Seeds treated with and without GR24 (10⁻⁶ M) were included as positive and negative controls.

5. Hydroponic culture of cucumber and collection of root exudate

The cucumber seeds were germinated on moistened vermiculite in plastic containers (28.5×23.5×11 cm, W×L×H) for 14 days at 23–27°C under natural daylight conditions. The plants were watered with tap water as required. Seedlings were transferred to a larger container (53.5×33.5×14 cm, W×L×H) containing 20 L of tap water. Each container containing about 50 seedlings was placed in a growth room maintained at 23–27°C under natural daylight conditions. Root exudates released into the culture medium were adsorbed on activated charcoal (4 g×2 for 20 L) using two water circulation pumps.^5,18) The plants were grown for 5 weeks and the culture medium and activated charcoal were exchanged every 3–4 days. The root exudates absorbed on charcoal (8 g) were eluted with acetone (400 mL). After evaporation of the acetone in vacuo, the aqueous residue (ca. 70 mL) was extracted with EtOAc (3×70 mL). The EtOAc extracts were combined, washed with 0.2 M K₂HPO₄ (100 mL, pH 8.3), dried over anhydrous MgSO₄, and concentrated in vacuo. The concentrated samples were kept at 4°C until use.

6. Isolation of 7α- and 7β-hydroxyorobanchyl acetate

The crude EtOAc extract (190.7 mg) collected over 5 weeks from cucumber seedlings grown hydroponically was subjected to silica gel CC (40 g) with stepwise elution of n-hexane–EtOAc (100 : 0–0 : 100, v/v, 10% step) to give fractions 1–11. Fractions 8 and 9 (70% and 80% EtOAc, respectively) containing two novel SLs were combined (13.35 mg) and subjected to silica gel CC (6 g) using n-hexane–EtOAc (40 : 60, v/v) as eluting solvent system. Fractions were collected every 10 mL. Fractions 17–19 and fractions 21–25 were found to contain novel germination stimulant 1 and 2, respectively, by LC-MS/MS and GC-MS analysis. Fractions 17–19 were combined (2.17 mg) and was purified by HPLC on an ODS column (Mightysil RP-18, 4.6×250 mm, 5 µm; Kanto Chemicals, Japan) with an MeCN/H₂O gradient system (10 : 90 to 60 : 40 over 50 min) as the eluent at a flow rate of 0.8 mL/min. The active fraction eluted as a single peak at 21.4 min (detection at 238 nm) was collected. This fraction was further purified by isocratic (70% MeCN/H₂O) HPLC on a Develosil ODS-CN column (4.6×250 mm, 5 µm; Nomura Chemicals, Japan) at a flow rate of 0.8 mL/min to give 7α-hydroxyorobanchyl acetate (1, 0.83 mg, R_t 27.7 min, detection at 238 nm). Fractions 21–25 of the second silica gel CC were combined (1.10 mg) and purified in a similar manner to give 7β-hydroxyorobanchyl acetate (2, 0.31 mg) with R_t 20.1 min in the ODS-HPLC and R_t 26.2 min in the ODS-CN HPLC (detection at 238 nm).

7α-Hydroxyorobanchyl acetate (1) CD (c 0.0010, MeCN) λ_max (Δε) nm: 218 (76.10), 254 (−5.61); GC-MS m/z: 404 (1) M⁺, 362 (2), 344 (4), 247 (22), 97 (100); HR-TOF-MS m/z (M+H⁺): Calcd. for C₂₁H₂₅O₈: 405.1549, Found: 405.1564. ¹H and ¹³C NMR spectroscopic data are shown in Table 1 and Supplemental Figs. S1–S3.

7β-Hydroxyorobanchyl acetate (2) CD (c 0.0005, MeCN) λ_max (Δε) nm: 218 (38.08), 254 (−4.11); GC-MS m/z: 404 (1) M⁺, 362 (2), 344 (5), 247 (23), 97 (100); HR-TOF-MS m/z (M+H⁺): Calcd. for C₂₁H₂₅O₈: 405.1549, Found: 405.1542. ¹H and ¹³C NMR spectroscopic data are shown in Table 1 and Supplemental Figs. S4–S6.

Table 1. NMR spectral data for compounds 1 and 2 (CDCl₃)

Position	1				2
Position	δ_H (mult., J Hz)	δ_C	HMBC	NOE	δ_H (mult., J Hz)	δ_C	HMBC	NOE
1
2		169.7				169.8
3		110.1				110.3
3a	3.48 (ddd, 7.3, 2.4, 1.9)	45.8	C-3, 4	H-8b	3.50 (ddd, 7.3, 2.4, 1.8)	45.4	C-3, 4	H-8b
4	5.72 (bs)	82.2	C-3, 8a		5.75 (bs)	82.5	C-3, 8a
4a		139.4				139.3
5	1.97–2.11 (m)	21.8	C-4a, 7		1.99–2.15 (m)	19.7	C-4a, 7
6	1.78–1.85 (m)	26.4	C-7, 9		1.77–1.95 (m)	25.4	C-7, 9
7	3.54 (dd, 8.2, 3.2)	75.3		H-9	3.62 (bd, 5.0)	74.8		H-10
8		37.3				37.3
8a		145.7				144.4
8b	5.60 (bd, 7.3)	85.3		H-3a, 9	5.60 (bd, 7.3)	85.7		H-3a, 9
9	1.24 (s)	21.0	C-7, 8, 8a, 10	H-7, 8b	1.21 (s)	21.4	C-7, 8, 8a, 10	H-8b
10	1.12 (s)	24.1	C-7, 8, 8a, 9		1.16 (s)	27.2	C-7, 8, 8a, 9	H-7
1′
2′	6.16 (t, 1.4)	99.8	C-6′	H-6′	6.16 (t, 1.4)	99.8	C-6′	H-6′
3′	6.95 (t, 1.4)	140.8		H-2′	6.95 (t, 1.4)	140.8		H-7′
4′		136.3				136.2
5′		170.2				170.2
6′	7.48 (d, 2.3)	150.8	C-2, 2′, 3, 3a	H-2′	7.48 (d, 2.4)	150.8	C-2, 2′, 3, 3a	H-2′
7′	2.03 (t, 1.4)	10.8	C-3′, 4′, 5′		2.03 (t, 1.4)	10.8	C-3′, 4′, 5′
1″		170.3				170.4
2″	2.04 (s)	20.7			2.04 (s)	21.0

Results and Discussion

1. Characterization of strigolactones produced by cucumber

Cucumber plants were grown hydroponically and root exudate was collected in a manner similar to that described previously.²¹⁾ The root exudate was subjected to solvent partitioning to afford a neutral EtOAc fraction. One ten-thousandth of this crude extract was analyzed by LC-MS/MS operated in the ESI positive mode. Four known SLs, 7-oxoorobanchyl acetate (3), orobanchyl acetate (5), orobanchol (6), and 4-deoxyorobanchol (7) were detected by monitoring the transitions m/z 425–268, 411–254, 369–272, and 353–256, respectively (Fig. 2A). The identities of these SLs were confirmed with cochromatography in LC-MS/MS using authentic SL standards. One hundred-thousandth of the crude extract was subjected to RP-HPLC operated under the same conditions as for LC-MS/MS analyses and the fractions collected every minute were examined for O. minor seed germination stimulation. The distribution of germination stimulation activities toward O. minor seeds suggested that the root exudates contained several novel germination stimulants including hydroxyorobanchyl acetate isomers in addition to these known SLs (Fig. 2B). In Fig. 2B, 4-deoxyorbanchol was eluted slightly earlier as compared to that in Fig. 2A.

Fig. 2. LC-MS/MS (MRM) chromatogram of cucumber root exudate (A). The monitoring transitions m/z 425–268, 411–254, 369–272, and 353–256 are for 7-oxoorobanchyl acetate (3), orobanchyl acetate (5), orobanchol (6), and 4-deoxyorobanchol (7), respectively. Distribution of germination stimulation activity of the cucumber root exudate after RP-HPLC (B). The fractions collected every minute were examined for O. minor seed germination stimulation.

2. Isolation and structural determination of 7α-hydroxyorobanchyl acetate (1) and 7β-hydroxyorobanchyl acetate (2)

The crude extract was first purified by a silica gel CC eluted with n-hexane–EtOAc. The germination stimulation activities on O. minor were eluted in the 40–90% EtOAc fractions. Six known SLs were detected from the 40% EtOAc fraction (4-deoxyorobanchol (7) and orobanchyl acetate (5)), the 50–60% EtOAc fractions (orobanchyl acetate (5), orobanchol (6), and 7-oxoorobanchyl acetate (3)) and the 90% EtOAc fraction (7-oxoorobanchol (4)). In addition to these SLs, the 70–80% EtOAc fractions were found to contain two novel SLs by LC-MS/MS analysis. The 70–80% EtOAc fractions were combined and subjected to silica gel CC and RP-HPLCs to afford pure compounds 1 and 2.

The HR-TOF-MS analyses of compounds 1 and 2 indicated that their molecular formulae were both C₂₁H₂₄O₈. The sodium adduct ion at m/z 427 (M+Na)⁺ in ESI-MS and the molecular ion at m/z 404 (M)⁺ in GC-MS also supported this molecular formula. The ¹H and ¹³C NMR spectra of compounds 1 and 2 were similar to those of orobanchyl acetate (5)^23,24) and especially to those of 7-oxoorobanchyl acetate (3),¹⁸⁾ indicating that these molecules contained the common structural feature for the known SLs.¹⁶⁾ The 2D NMR (HMQC, HMBC, NOE and COSY) data also confirmed these assignments (Table 1, Supplemental Figs. S1–S6).

The doublet of doublet signal at 3.54 ppm (J=8.2, 3.2 Hz) of compound 1 indicated the presence of a hydroxyl group at C-7. The absence of methylene proton signals at C-7 also supported the presence of a 7-hydroxyl group. The H-7 signal of compound 1 shows NOE correlations with H-8b (weak) and 9-Me (strong) and the 9-Me signal with H-7 and H-8b, indicating that the relative stereochemistry of the 7-hydroxyl group in compound 1 is cis to the C ring. By contrast, the NOE correlation between H-7 and 10-Me in compound 2 demonstrates that the relative stereochemistry of the hydroxyl group is opposite to that in compound 1. Therefore, compounds 1 and 2 are diastereomers with stereochemistry different from the 7-hydroxyl group. The CD spectra of compounds 1 and 2 showed a positive and negative Cotton effect at around 218 nm and 254 nm, respectively, which were similar to that of orobanchol-type SLs suggesting that compounds 1 and 2 have a 2′(R) configuration carrying an α-oriented C-ring (Fig. 1).²⁵⁾ Accordingly, compounds 1 and 2 were determined to be 7α-hydroxyorobanchyl acetate and 7β-hydroxyorobanchyl acetate, respectively (Supplemental Fig. S7).

3. Detection of 7α-hydroxyorobanchol (8), 7β-hydroxyorobanchol (9), and other minor strigolactones

In the 90% EtOAc fraction of the first silica gel CC, which was moderately active in O. minor seed germination assay, two novel germination stimulants were detected by LC-MS/MS monitoring of the transition of m/z 385–288, indicating that these were hydroxyorobanchol isomers. Therefore this fraction was subjected to purification by RP-HPLCs, but we could obtain only trace amounts (<0.1 µg) of an inseparable mixture of two isomeric compounds that decomposed gradually, even in solutions with aprotic organic solvents. The LC-MS and GC-MS analytical data of these compounds strongly suggested that these compounds are 7α-hydroxyorobanchol (8) and 7β-hydroxyorobanchol (9) (Supplemental Figs. S8 and S9). Indeed, acetylation of these compounds afforded monoacetyl esters that showed the same retention times and spectra in the LC-MS/MS and GC-MS analytical data as those of compounds 1 and 2 (data not shown).

In addition to these SLs, three SL-like germination stimulants were detected by LC-MS/MS. In the mass spectra, they showed fragmentation patterns typical for SLs, such as a base peak at m/z 97 in GC-MS. The molecular weights of two of them were found to be 374 and that of the other to be 390. Purification and structural determination of these compounds are now in progress.

4. Germination stimulation activities of 7α-hydroxyorobanchyl acetate (1) and 7β-hydroxyorobanchyl acetate (2) toward O. minor and P. ramosa seeds

The germination stimulation activities of 7α-hydroxyorobanchyl acetate (1), 7β-hydroxyorobanchyl acetate (2), 7-oxoorobanchyl acetate (3) and orobanchyl acetate (5) toward O. minor and P. ramosa seeds are shown in Fig. 3. All SLs induced similar levels of high germination (ca. 80%) of O. minor seeds at 0.1 nM. These results indicate that the introduction of a carbonyl or hydroxyl group at C-7 does not affect the germination activity toward O. minor seeds. By contrast, 7β-hydroxyorobanchyl acetate (2) was found to be a highly potent germination stimulant for P. ramosa seeds, inducing more than 50% germination at 10 pM, suggesting that the β-hydroxyl group at C-7 may be involved in the interaction between the stimulant and its receptor site in the seed of this parasitic weed.

Fig. 3. Germination stimulation activities of 7α-hydroxyorobanchyl acetate (1), 7β-hydroxyorobanchyl acetate (2), 7-oxoorobanchyl acetate (3) and orobanchyl acetate (5), toward O. minor and P. ramosa seeds. Data are presented as means±standard errors (n=3).

Conclusion

This study demonstrates that cucumber plants exude a mixture of at least 12 germination stimulants—most likely SLs. Since all plant species examined have been shown to produce and release mixtures of SLs, it is intriguing to understand whether root parasitic weed seeds, AM fungi, and other beneficial or pathogenic microorganisms need special cocktails of SLs for their host’s recognition. Furthermore, recently identified carlactone-related germination stimulants²⁶⁾ may also contribute to the interaction between plants and other organisms in the rhizosphere. Further studies are needed to clarify the roles of these plant-derived rhizosphere-signaling chemicals in the establishment and maintenance of underground plant, microbial, and faunal communities.

Acknowledgment

This work was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry and by the special grant for the UU-COE from Utsunomiya University. PK is supported by Japanese Government (Monbukagakusho: MEXT) Scholarship.

References

1) D. M. Joel, J. C. Steffens and D. E. Matthews: “Seed Development and Germination,” eds. by J. Kigel and G. Galili, Marcel Dekker, Inc., New York, pp. 567–597, 1995.
2) X. Xie, K. Yoneyama and K. Yoneyama: Annu. Rev. Phytopathol. 48, 93–117 (2010).
3) H. J. Bouwmeester, R. Matusova, S. Zhongkui and M. H. Beale: Curr. Opin. Plant Biol. 6, 358–364 (2003).
4) H. I. Kim, X. Xie, H. S. Kim, J. C. Chun, K. Yoneyama, T. Nomura, Y. Takeuchi and K. Yoneyama: J. Pestic. Sci. 35, 344–347 (2010).
5) K. Akiyama, K.-i. Matsuzaki and H. Hayashi: Nature 435, 824–827 (2005).
6) V. Gomez-Roldan, S. Fermas, P. B. Brewer, V. Puech-Pagès, E. A. Dun, J.-P. Pillot, F. Letisse, R. Matusova, S. Danoun, J.-C. Portais, H. Bouwmeester, G. Bécard, C. A. Beveridge, C. Rameau and S. F. Rochange: Nature 455, 189–194 (2008).
7) M. Umehara, A. Hanada, S. Yoshida, K. Akiyama, T. Arite, N. Takeda-Kamiya, H. Magome, Y. Kamiya, K. Shirasu, K. Yoneyama, J. Kyozuka and S. Yamaguchi: Nature 455, 195–200 (2008).
8) Y. Kapulnik, P.-M. Delaux, N. Resnick, E. Mayzlish-Gati, S. Wininger, C. Bhattacharya, N. Séjalon-Delmas, J.-P. Combier, G. Bécard, E. Belausov, T. Beeckman, E. Dor, J. Hershenhorn and H. Koltai: Planta 233, 209–216 (2011).
9) C. Ruyter-Spira, W. Kohlen, T. Charnikhova, A. van Zeijl, L. van Bezouwen, N. de Ruijter, C. Cardoso, J. A. Lopez-Raez, R. Matusova, R. Bours, F. Verstappen and H. Bouwmeester: Plant Physiol. 155, 721–734 (2011).
10) H. Shen, P. Luong and E. Huq: Plant Physiol. 145, 1471–1483 (2007).
11) J. Agusti, S. Herold, M. Schwarz, P. Sanchez, K. Ljung, E. A. Dun, P. B. Brewer, C. A. Beveridge, T. Sieberer, E. M. Sehr and T. Greb: Proc. Natl. Acad. Sci. U.S.A. 108, 20242–20247 (2011).
12) K. C. Snowden, A. J. Simkin, B. J. Janssen, K. R. Templeton, H. M. Loucas, J. L. Simons, S. Karunairetnam, A. P. Gleave, D. G. Clark and H. J. Klee: Plant Cell 17, 746–759 (2005).
13) S. Toh, Y. Kamiya, N. Kawakami, E. Nambara, P. McCourt and Y. Tsuchiya: Plant Cell Physiol. 53, 107–117 (2012).
14) X. Xie, K. Yoneyama, T. Kisugi, K. Uchida, S. Ito, K. Akiyama, H. Hayashi, T. Yokota, T. Nomura and K. Yoneyama: Mol. Plant 6, 153–163 (2013).
15) K. Yoneyama, T. Kisugi, X. Xie and K. Yoneyama: “Molecular Microbial Ecology of The Rhizosphere,” ed. by F. J. de Bruijn, Wiley-Blackwell Publishers, pp. 373–379, 2013.
16) K. Yoneyama, X. Xie and K. Yoneyama: “Handbook of Natural Products,” eds. by K. G. Ranawat and J. M. Merillon, Springer-Verlag GmbH, Heidelberg, pp. 3583–3604, 2013.
17) A. Alder, M. Jamil, M. Marzorati, M. Bruno, M. Vermathen, P. Bigler, S. Ghisla, H. Bouwmeester, P. Beyer and S. Al-Babili: Science 335, 1348–1351 (2012).
18) X. Xie, K. Yoneyama, J.-y. Kurita, Y. Harada, Y. Yamada, Y. Takeuchi and K. Yoneyama: Biosci. Biotechnol. Biochem. 73, 1367–1370 (2009).
19) T. Yokota, H. Sakai, K. Okuno, K. Yoneyama and Y. Takeuchi: Phytochemistry 49, 1967–1973 (1998).
20) T. Kisugi, X. Xie, H. I. Kim, K. Yoneyama, A. Sado, K. Akiyama, H. Hayashi, K. Uchida, T. Yokota, T. Nomura and K. Yoneyama: Phytochemistry 87, 60–64 (2013).
21) K. Yoneyama, K. Yoneyama, Y. Takeuchi and H. Sekimoto: Planta 225, 1031–1038 (2007).
22) J. Matsui, T. Yokota, M. Bando, Y. Takeuchi and K. Mori: Eur. J. Org. Chem. 1999, 2201–2210 (1999).
23) X. Xie, K. Yoneyama, D. Kusumoto, Y. Yamada, T. Yokota, Y. Takeuchi and K. Yoneyama: Phytochemistry 69, 427–431 (2008).
24) H. Matsuura, K. Ohashi, H. Sasako, N. Tagawa, Y. Takano, Y. Ioka, K. Nabeta and T. Yoshihara: Plant Growth Regul. 54, 31–36 (2008).
25) P. Welzel, S. Röhrig and Z. Milkova: Chem. Commun. (Camb.), 2017–2022 (1999).
26) H. I. Kim, T. Kisugi, P. Khetkam, X. Xie, K. Yoneyama, K. Uchida, T. Yokota, T. Nomura, C. S. P. McErlean and K. Yoneyama: Phytochemistry 103, 85–88 (2014).

Corresponding author

Register with J-STAGE for free!